Movement of sea urchin sperm flagella

The motion of the sea urchin sperm flagellum was analyzed from high-speed cinemicrographs. At all locations on the flagellum the transversal motion and the curvature were found to vary sinusoidally in time. The curvatures of the flagella increase strongly near the proximal junction. Two sperm are described in transient from rest to normal motion. The full wave motion developed in both sperm within 40 ms.     

Calcium sensors in sea urchin sperm flagella

The asymmetry of ATP-reactivated flagellar bending waves of Triton-demembrated sea urchin spermatozoa has been measured over a range of free Ca2+ ion concentrations from 10(-9) to 10(-4) M. Detailed examination of the gradual response of asymmetry to Ca2+ ion concentration over this wide range indicates the presence of two Ca2+ sensors. A high-affinity sensor operates at Ca2+ concentrations near 10(-7.5) M. A lower-affinity sensor operates at Ca2+ concentrations above 10(-6) M, in the typical range for calmodulin-mediated responses. Incubation of demembranated sperm flagella at high Ca2+ concentrations to release calmodulin is required to enable these Ca2+ responses to be observed. This treatment also causes a decrease in the apparent affinity of the flagella for calmodulin, as determined by measuring the increase in asymmetry in response to addition of exogenous calmodulin at low Ca2+ concentration.     

Calcium-induced asymmetrical beating of triton-demembranated sea urchin sperm flagella

Asymmetrical bending waves can be obtained by reactivating demembranated sea urchin spermatozoa at high Ca2+ concentrations. Moving-film flash photography shows that asymmetrical flagellar bending waves are associated with premature termination of the growth of the bends in one direction (the reverse bends) while the bends in the opposite direction (the principal bends) grow for one full beat cycle, and with unequal rates of growth of principal and reverse bends. The relative proportions of these two components of asymmetry are highly variable. The increased angle in the principal bend is compensated by a decreased angle in the reverse bend, so that there is no change in mean bend angle; the wavelength and beat frequency are also independent of the degree of asymmetry. This new information is still insufficient to identify a particular mechanism for Ca2+-induced asymmetry. When a developing bend stops growing before initiation of growth of a new bend in the same direction, a modification of the sliding between tubules in the distal portion of the flagellum is required. This modification can be described as a superposition of synchronous sliding on the metachronous sliding associated with propagating bending waves. Synchronous sliding is particularly evident in highly asymmetrical flagella, but is probably not the cause of asymmetry. The control of metachronous sliding appears to be unaffected by the superposition of synchronous sliding.     

Inhibition and relaxation of sea urchin sperm flagella by vanadate

Direct measurements of the stiffness (elastic bending resistance) of demembranated sera urchin sperm flagella were made in the presence of MgATP2- and vanadate. Under these conditions, the flagellum is in a relaxed state, with a stiffness of approximately 0.9 x 10(-21) N m2, which is approximately 5% of the stiffness obtained in the rigor state in the absence of MgATP2-. MgADP- dose not substitute for MgATP2- in producing relaxed state. A progressive inhibition of movement is observed after addition of MgATP2- to flagella preincubated with vanadate, in which new bend generation, propagation, and relaxation by straightening are distinguished, depending on the ratio of MgATP2- and vanadate. At appropriate concentrations of vanadate, increase of the velocity of bend propagation is observed at a very low concentration of MgATP2- that is not enough to induce spontaneous beating. Vanadate enhances competitive inhibition of beat frequency by MgADP- but not by ADP3-, ATP4-, or Pi. These observations, and the uncompetitive inhibition of beat frequency by vanadate, indicate that vanadate can only bind to dynein-nucleotide complexes induced by MgATP2- and MgADP-. The state accessible by MgATP2- binding must be a state in which the cross-bridges are detached and the flagellum is relaxed. The state accessible by MgADP- binding must be a cross-bridged state. Bound vanadate prevents the transition between these two states. Inhibition and relaxation by banadate in the presence of MgATP2- results from the specific affinity of vanadate for a state in which nucleotide is bound, rather than a specific affinity for the deteched state.     

Algebraic expressions for the waveforms of sea urchin sperm flagella

The waveforms of live sea urchin sperm flagella were digitized with video-digitizing apparatus. The flagellar waveforms were expressed by the coordinates of 20 points spaced 2 micron apart along the flagella. The waveforms were condensed into simple algebraic expression with four parameters. Each of these parameters showed a systematic variation with the flagellar frequency. These average trends of the parameters made it possible to define an average, idealized, waveshape as a function of the flagella frequency, in the range 8-80 Hz.     

Polypeptide subunits of dynein 1 from sea urchin sperm flagella

A high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to show the presence, in both whole sperm and isolated flagellar axonemes, of eight polypeptides migrating in the 300,000–350,000 molecular weight range characteristic of the heavy chains of dynein ATPase. Previously, only five such chains have been discernible. Extraction of isolated axonemes for 10 min at 4°C with a solution containing 0.6 M NaCl, pH 7, releases a mixture of particles that separate, in sucrose density gradient centrifugation, into a major peak, dynein 1 ATPase, sedimenting at 21 S and a minor peak at 12–14S. The polypeptide compositions of these two peaks are different. The dynein 1 peak, which contains most of the protein on the gradient, contains approximately equal quantities of two closely migrating heavy chains, with a small amount of a third, more slowly migrating chain; no other heavy chains appear in this peak. Two groups of smaller polypeptides (three intermediate chains, within the apparent molecular weight range 76,000–122,000 and four newly discovered light chains, within the apparent molecular weight range 14,000–24,000) cosediment with the 21 S peak. The heavy chain composition of the 12–14S peak is more complex, all eight heavy chains occurring in approximately the same ratios as occur in intact axonemes.     

Motility of triton-demembranated sea urchin sperm flagella during digestion by trypsin

The survival curves for a population of reactivated spermatozoa exposed to digestion by trypsin indicate that a large number of trypsin-sensitive targets must be digested before the flagellum disintegrates. Changes in flagellar movement during trypsin digestion can be very small, especially when the spermatozoa are reactivated at 0.25 M KCl. They are not the changes which would be expected if elastic resistance of the trypsin-sensitive structures responsible for maintaining the integrity of the axoneme is a significant determinant of flagellar bend amplitude. By carrying out trypsin digestion under a variety of conditions, at least six distinct effects of trypsin digestion on parameters of flagellar movement have been detected. These include a gradual increase in the rate of sliding between tubules, gradual and abrupt changes in beat frequency accompanied by reciprocal decreases in bend angle, changes in the symmetry and planarity of bending, and selective interference with mechanisms for bend initiation and bend propagation.     

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.     

Mechanochemical coupling in the relaxation of rigor-wave sea urchin sperm flagella

The relaxation (straightening) of flagellar rigor waves, which is known to be induced by micromolar ATP concentrations was investigated with respect to its dependence on the binding and hydrolysis of ATP. Flagellar rigor waves were formed by the dilution of demembranated, reactivated sea urchin (Lytechinus pictus) spermatozoa into ATP-free buffer. Relaxation in response to nucleotide was quantitated by measuring theta, the mean flagellar bend angle per sperm; this novel assay permitted determination of the rate of relaxation. It was found that (a) the rate of flagellar relaxation induced by 4 X 10(-6) M ATP was inhibited 80% by vanadate concentrations of 3 X 10(-6) M and above; (b) of 16 hydrolyzable and nonhydrolyzable nucleotide di-, tri-, and tetraphosphates tested, only three, each of which was hydrolyzed by the flagellar axonemal ATPase activity (ATP, dATP, and epsilon-ATP) were also capable of effecting relaxation; (c) several hundred ATP molecules were estimated to be hydrolyzed by each dynein of ATP hydrolysis, which defines the efficiency of ATP utilization, increased 30-fold as the ATP relaxation depends on ATP hydrolysis; (b) because it depends on ATP hydrolysis, flagellar relaxation is an inappropriate model system for investigating the role of ATP binding in the mechanochemical cycle of dynein; and (c) the efficiency of mechanochemical coupling in flagellar motility is an ATP-dependent phenomenon. A general model of relaxation is proposed based on active microtubule sliding.     

A new hyperpolarization-activated, cyclic nucleotide-gated channel from sea urchin sperm flagella

A sea urchin sperm flagellar hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel is known (SpHCN1) that is modulated by cAMP. Here, we describe a second flagellar HCN channel (SpHCN2) cloned from the same sea urchin species. SpHCN2 is 638 amino acids compared to 767 for SpHCN1. SpHCN2 has all the domains of an HCN channel, including six transmembrane segments (S1-S6), the ion pore, and the cyclic nucleotide-binding domain. The two full-length proteins are 33% identical and 51% similar. The six transmembrane segments vary from 46-79% identity. S4, which is the voltage sensor, is 79% identical between the two proteins. The ion selectivity filter sequence is GYG in the ion pore of SpHCN1 and GFG in SpHCN2. By sequence, SpHCN2 is 73.5kDa, but it migrates on SDS-PAGE at 64kDa. Western immunoblots show localization to flagella, which is confirmed by immunofluorescence. A neighbor-joining tree shows that SpHCN2 is basal to all known HCN channels. SpHCN2 might be the simplest pacemaker channel yet discovered.     

Two heavy chains of 21S dynein from sea urchin sperm flagella

The biochemical properties of 21S dynein derived from sea urchin sperm flagella and of its components dissociated by low salt treatment were studied. SDS-urea gel electrophoresis and two-dimensional gel electrophoresis showed that the 21S dynein preparation contains two distinct heavy chains. These two heavy chains, termed A alpha and A beta, had apparently the same molecular weight of 500,000 but showed different mobilities on SDS-urea gels. The isoelectric points of A alpha and A beta heavy chains were 5.7 and 5.2, respectively, in the presence of urea. Proteolytic digestion patterns of these two heavy chains were clearly different, but the amino acid compositions were similar. Low salt treatment and sucrose density gradient centrifugation could partially separate the components of 21S dynein into two fractions: the one with larger sedimentation coefficient contained the A alpha heavy chain, and the other with smaller sedimentation coefficient contained the A beta heavy chain and three intermediate chains. These two fractions showed distinctly different kinetic properties, and thus may play different roles in dynein-microtubule interaction.     

Conformational change in the outer doublet microtubules from sea urchin sperm flagella

Dark-field microscopy with a high-powered light source revealed that the outer doublet microtubules (DMTs) from sea urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella assume helically coiled configurations (Miki-Noumura, T., and R. Kamiya. 1976. Exp. Cell Res. 97: 451.). We report here that the DMTs change shape when the pH or Ca-ion concentration is changed. The DMTs assumed a left-handed helical shape with a diameter of 3.7 +/- 0.5 micron and a pitch of 2.8 +/- 0.7 micron at pH 7.4 in the presence of 0.1 mM CaCl2, 1 mM MgSO4, and 10 mM Tris-HCl. When the pH was raised to 8.3, the helical diameter and pitch decreased to 2.1 +/- 0.1 micron and 1.3 +/- 0.3 micron, respectively. This transformation was a rapid and reversible process and was completed within 1 min. Between pH 7.2 and 8.3, the DMTs assumed intermediate shapes. When the Ca-ion concentration was depleted with EGTA, the helical structure became significantly larger in both pitch and diameter. For instance, the diameter was 3.8 +/- 0.4 micron at pH 8.3 in the presence of 1 mM EGTA and 2 mM MgSO4. Using a Ca-buffer system, we obtained results which suggested that this Ca-induced transformation took place at a Ca concentration of approximately 10(-7) M. These results were highly reproducible. The conformational changes in the DMT may play some role in the bending wave form of flagellar movement.     

Effects of antibodies against tubulin on the movement of reactivated sea urchin sperm flagella

Antibodies binding to sea urchin flagellar outer-doublet tubulin have been isolated from rabbit sera by tubulin-affinity chromatography employing electrophoretically purified tubulin as the immobilized substrate. This procedure provides "induced" antitubulin antibody from immune sera and "spontaneous" antitubulin antibody from preimmune sera. These antitubulins were characterized in terms of their specificity, ability to bind to sea urchin axonemes, and effects on the motility of reactivated spermatozoa. Induced antitubulin antibody specifically reduced the bend angle and symmetry of the movement of demembranated reactivated spermatozoa without affecting the beat frequency. At identical concentrations, spontaneous antitubulin had no effect on motility. Affinity-purified induced antitubulins from three other rabbits all gave specific bend-angle inhibition, whereas their corresponding spontaneous antitubulins had no effect on the flagellar movement. The effects of antitubulin on microtubule sliding were examined by observing the sliding disintegration of elastase-digested axonemes induced by MgATP2+-. Affinity-purified induced antitubulin antibody, in quantities sufficient to completely paralyze reactivated flagella, did not inhibit microtubule sliding. The amplitude-inhibiting effect of induced antitubulin on reactivated spermatozoa may be caused by action on a mechanism responsible for controlling flagellar bending rather than by interference with the active sliding process. This is the first report of an antitubulin antibody having an inhibitory activity on microtubule-associated movement.     

Dynein 2. A new adenosine triphosphatase from sea urchin sperm flagella.

A new ATPase electrophoretically and immunologically distinct from the dynein ATPase studied previously has been solublized and purified from sea urchin sperm flagella. This ATPase has properties similar to those of dynein ATPase. Therefore, we propose that the two ATPases be considered as dynein isoenzymes, with previously studied dynein being known as dynein 1, and the newly discovered ATPase as dynein 2. Some physicochemical and enzymatic properties of dynein 2 have been determined. The molecular weight calculated from the sedimentation coefficient (12.3 "/- 1 S) and Stokes radius (12.8 "/- 0.4 nm) is 690,000 +/- 70,000. The molecular weight of the high molecular weight subunit of dynein 2 has been determined to be 325,000 +/- 40,000 by Na dodecyl-SO4-polyacrylamide gel electrophoresis. The enzymatic properties of dynein 1 and dynein 2 are similar in substrate specificity, pH optimum, and Mg2+ requirement for ATPase activity, but they differ in their Michaelis constant and in their dependence of ATPase activity upon salt concentration. Digestion of dynein 2 with trypsin yields an ATPase-containing protein fragment, similar to Fragment A obtained from dynein 1. An antiserum prepared against Fragment A from dynein 1 did not precipitate dynein 2 or inhibit its ATPase activity.     

A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella.

Treatment of demembranated sea urchin sperm axonemes with an extraction solution containing 0.6 M NaCl, pH 7.0 for 10 min at 4 degrees C yields a solution of dynein 1 having a low, latent specific ATPase activity of about 0.25 mumol of Pi mg(-1) min(-1). Exposure of this dynein solution to 0.1% Triton-X-100 for 10 min at 25 degrees C causes an increase in its ATPase activity to about 3 mumol of Pi mg(-1) min(-1). A similar activation can be obtained by treating at 42 degrees C or by reacting with 60 mol of p-chloromercuribenzene sulfonate/10(6) g of protein. The effects of these activating procedures are not additive, suggesting that they lead to a common activated state. Purification of the latent activity dynein 1 by sucrose density gradient centrifugation yields a monodisperse preparation sedimenting at 21 S, and having a molecular weight of 1,250,000 as determined by sedimentation diffusion and sedimentation equilibrium. Activation of the latent dynein 1 with Triton X-100 converts it to a form sedimenting at 10 to 14 S. The 21 S dynein is also converted to a 10 S form by dialysis against 5 mM imidazole/NaOH buffer, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, pH 7, although in this case, the ATPase activity is increased only about 3-fold, with another 3-fold activation being obtainable upon subsequent treatment with Triton X-100. The 21 S latent form of dynein 1 may represent the intact dynein arms that form moving cross-bridges and generate active sliding between adjacent doublet tubules of the flagellar axoneme. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate suggests a model in which the 21 S dynein 1 particle is composed of three subunits of about 330,000 daltons and one of each of three medium weight subunits of 126,000, 95,000, and 77,000 daltons. When latent dynein 1 is added back to NaCl-extracted axonemes in the presence of 0.15 M NaCl, it recombines stoichiometrically and restores the arms on the doublet tubules with a 6-fold activation of its ATPase activity measured in the absence of KCl.     

The force-velocity relationship for microtubule sliding in demembranated sperm flagella of the sea urchin

We studied the relationship between the force and velocity of microtubule sliding in demembranated sperm flagella of the sea urchin, Hemicentrotus pulcherrimus, under auxotonic conditions following a quick release of the tension between sliding microtubules. The shape of the force-velocity curve was independent of the concentration of Mg-ATP over the range of 3.7 to 350 microM and appeared either linear or was the reverse of the hyperbolic curve seen for muscle. The power, calculated as the product of velocity and force, passed through a peak at c. 0.7 Fmax (the maximal isometric force). Thus, the maximal power is attained at a larger relative load than in muscle. The sliding velocity at 0.1 Fmax showed a hyperbolic dependence on Mg-ATP concentration, with a Km of 210 microM and a Vmax of 19 micron.sec-1. The maximal force did not significantly change over the Mg-ATP concentration range of 3.7 to 350 microM. These results are discussed in terms of a crossbridge model similar to the one originally proposed by Huxley. It is suggested that the dynein crossbridge cycle is characterized by a relatively rapid rate of attachment and a relatively slow rate of detachment.     

Localization of tektin filaments in microtubules of sea urchin sperm flagella by immunoelectron microscopy

Extraction of doublet microtubules from the sperm flagella of the sea urchin Strongylocentrotus purpuratus with sarkosyl (0.5%)-urea (2.5 M) yields a highly pure preparation of "tektin" filaments that we have previously shown to resemble intermediate filament proteins. They form filaments 2-3 nm in diameter as seen by negative stain electron microscopy and are composed of approximately equal amounts of three polypeptide bands with apparent molecular weights of 47,000, 51,000, and 55,000, as determined by SDS PAGE. We prepared antibodies to this set of proteins to localize them in the doublet microtubules of S. purpuratus and other species. Tektins and tubulin were antigenically distinct when tested by immunoblotting with affinity-purified antitektin and antitubulin antibodies. Fixed sperm or axonemes from several different species of sea urchin showed immunofluorescent staining with antitektin antibodies. We also used antibodies coupled to gold spheres to localize the proteins by electron microscopy. Whereas a monoclonal antitubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol. 93:576-582) decorates intact microtubules along their lengths, antitektins labeled only the ends of intact microtubules and sarkosyl-insoluble ribbons. However, if microtubules and ribbons attached to electron microscope grids were first extracted with sarkosyl-urea, the tektin filaments that remain were decorated by antitektin antibodies throughout their length. These results suggest that tektins form integral filaments of flagellar microtubule walls, whose antigenic sites are normally masked, perhaps by the presence of tubulin around them.     

The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella

Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.     

External mechanical control of the timing of bend initiation in sea urchin sperm flagella

The movement parameters of a sea urchin sperm flagellum can be manipulated mechanically by applying various modes of periodic vibrations to the sperm head held by suction in the tip of a micropipette. The beat frequency of the flagellum readily synchronizes with the frequency of the externally imposed lateral vibration, and the plane of flagellar bending waves adapts itself to the plane of the pipette vibration (Gibbons et al., J. Cell Biol. 101:270a, 1985; Nature 325: 351-352, 1987). In this study, we observed the particular effects of external asymmetric forces on flagellar beating parameters by vibrating the micropipette holding the sperm head in a transverse sawtooth-like motion composed of a rapid effective stroke and a slower recovery stroke, while keeping the vibration frequency constant. The results demonstrate that the timing of bend initiation within the flagellar beat cycle can be controlled mechanically by changing the time point within the vibration cycle at which the micropipette changes its direction of motion. A switch in the sidedness of the asymmetric movement of the micropipette produces dramatic changes in the profiles of bend growth in the basal 5 microns of the flagellum but has almost no effect on the asymmetry or other parameters of bending in the mid- and distal regions of the flagellum. Our results suggest that elastic strain within the basal region of the flagellar structure may play a more significant role in the process of bend initiation than has been realized heretofore.